Ryanodine receptors (RyRs) are large intracellular Ca2+ channels that mobilize stored Ca2+ in cardiac and skeletal muscle to initiate muscle contraction. Recent studies have shown that Ca2+ mobilization by RyRs in airway smooth muscle (ASM) also plays a prominent role in airway constriction (bronchoconstriction). Moreover, exaggerated Ca2+ mobilization by these channels under inflammatory conditions may contribute to airway hyperresponsiveness. We previously reported that murine ASM expresses all three RyR isoforms. We have subsequently used a gene-silencing "knockdown" approach to determine each RyR isoform's role in bronchoconstriction provoked by cholinergic agonists, perhaps the most clinically relevant group of airway spasmogens. Our data indicate that type 2 RyR (RyR2) and then type 1 RyR (RyR1) are sequentially activated in ASM cells to mediate the dose-dependent Ca2+ responses provoked by cholinergic agonists. Therefore, the long-term objectives of this proposal are to: 1) determine the cellular pathways that sequentially activate of these two RyR isoforms in cholinergic agonist-stimulated ASM;and 2) determine whether at least one of these pathways is heightened by airway inflammation in vivo, thus may play a role in the pathogenesis of airway hyperresponsiveness. In Aim 1 we will test the hypothesis that RyR2 in ASM cells is first activated by a cellular pathway dependent on type 2 muscarinic (M2) receptor and an ADP- ribosyl cyclase CD38;but RyR1 is separately activated through type 3 muscarinic receptor as the level of cholinergic stimulation increases. In Aim 2 we will use a combination of knockdown, electrophysiology and Ca2+ imaging techniques to test the hypothesis that in ASM RyR1 is functionally coupled to a voltage- dependent L-type channel as in skeletal muscle;and a reciprocal activation between these two channels plays a role in mobilizing Ca2+ in carbachol-stimulated ASM cells. In Aim 3, we will study wild-type, M2 receptor-deficient and CD38-deficient mice to determine whether an upregulation of the proposed pathway that activates RyR2 plays a role in the airway hyperresponsiveness seen in two distinct in vivo models of airway inflammation. The completion of these Aims will improve our understanding of the cellular mechanisms that mediate the dose-dependent Ca2+ responses ASM provoked by cholinergic agonists and may identify therapeutic targets for respiratory diseases characterized by excessive bronchoconstriction.